Toxoplasmosis is the most common cause of focal central nervous system (CNS) disease in AIDS. AIDS patients who survive Toxoplasmosis exhibit significant neurological deficits. Moreover, latent Toxoplasma gondii [Toxoplasma] infection is strongly associated with worse neurocognitive functioning in HIV-positive patients. These CNS clinical manifestations originate from the reactivation of latent Toxoplasma cysts. There is no current therapy that can eradicate latent cysts or reduce the risk of reactivated infection in HIV-positive and in AIDS patients. Developing any therapy to reduce the disease burden of latent Toxoplasma infection has lagged because of major gaps in our biological knowledge and understanding of latent Toxoplasma infection. These gaps in knowledge have been driven because of the lack of any genetic model for latent infection that can be specifically applied to validate drug targets for latent infection, or to clearly distinguish a specific biological role for a parasite gene in latent infection. For example, we and others have deleted various genes encoding molecules secreted by Toxoplasma and reported major or complete defects in the ability of some of these mutants to establish latent infection in mice. Unfortunately today, the field cannot easily or clearly decipher whether the Toxoplasma genes that impact latent infection perform their biological functions during acute infection (which precedes latent infection), during the transition from acute to latent infection, or during the latent cyst stages. To eradicate latent cysts in HIV-positive and in AIDS patients, the field must identify and validate drug targets that can directly act on latent cysts to cause their disintegration and clearance. To validate these potential drug targets the field requires a genetic model that can turn off gene function after latent cysts have already developed. While these genetic models are currently available for acute Toxoplasma infection for which we do have some clinical treatments, these genetic models are not yet developed for latent cyst stages for which we do not have any clinical treatments. We propose to develop and validate a genetic model to control Toxoplasma gene expression in the latent cyst stages. The genetic model(s) developed in this high impact R21 project will be made broadly available to Toxoplasma researchers to accelerate the discovery of parasite genes that are essential for establishing and maintaining the latent cyst stages.